barcode amplicon sequence from Bacteroides fragilis p207 bar-seq library cultured in plain BHIS medium. 1 day of growth.replicate4
To assess barcode abundances, we followed the approach developed and described by Wetmore and colleagues [doi: 10.1128/mBio.00306-15].  Briefly, each cell pellet was resuspended in approximately 50 ul water.  Barcodes were amplified using Q5 polymerase (New England Biolabs) in 20 ul reaction volumes containing 1X Q5 reaction buffer, 1X GC enhancer, 0.8 U Q5 polymerase, 0.2 mM dNTP, 0.5 uM of each primer and 1 ul of resuspended cells.  Each reaction contained the Barseq_P1 forward primer and a unique indexed reverse primer, Barseq_P2_ITxxx, where the xxx identifies the index number.  Reactions were cycled as follows: 98 C for 4 min, 25X (98 C for 30 s, 55 C for 30 s, and 72 C for 30), 72 C for 5 min, 4C hold.  PCR products run on a 2% agarose gel to confirm amplification of a 190 bp product.  Aliquots (5 ul) of each reaction were pooled.  The pool was centrifuged for 2 min at 16,000g to pellet cell debris.  Then 90 ul of the mix was transferred to a fresh tube and cleaned with SPRIselect beads (Beckman Coulter) using a two-sided (1.2x-0.5X) selection. The amplified barcodes were sequenced using a 75-cycle MiniSeq High Output Reagent Kit (Illumina, FC-420-1001) on an Illumina MiniSeq.  To aid in clustering, sequencing runs were supplemented with 20-30% phiX DNA